Method for the detection of influenza A/B viruses

ABSTRACT

The invention concerns a method for the detection of an infection with influenza A and/or influenza B virus comprising the steps: i) obtaining a saliva sample, ii) preparing the saliva sample for a detection reaction and iii) detecting the influenza A and/or influenza B virus in the saliva sample. The invention in addition concerns a test kit for the detection of an infection by the influenza A and/or influenza B virus containing: i) a device for the collection of a saliva sample and ii) reagents and auxiliary agents for the detection of influenza A and/or influenza B viruses. Furthermore the invention concerns the use of saliva as a sample material for the detection of an infection with the influenza A and/or influenza B virus.

FIELD OF THE INVENTION

The invention concerns a method for the detection of influenza A/Bviruses, a corresponding test kit and the use of saliva as a samplematerial for the detection of influenza A/B viruses.

BACKGROUND OF THE INVENTION

Influenza is a frequently underestimated infectious disease which canresult in high morbidity and mortality rates especially in elderlypersons and in high-risk patients. Influenza A and/or influenza Bviruses (also abbreviated influenza A/B viruses in the following) areresponsible for genuine virus influenza which is contracted by several100 million persons worldwide each year. The influenza A and B virusesprimarily infect the nasopharyngeal and oropharyngeal cavities andinitially cause general respiratory symptoms in the affected persons.

It is not possible even for experienced medical professionals to veryreliably diagnose influenza solely on the basis of the patient'sclinical symptoms since other viruses which infect the nasal orpharyngeal cavity such as adenoviruses, parainfluenza viruses orrespiratory syncitial viruses (RS viruses) cause similar symptoms.

Due to the high medical importance of influenza infections (flu), almostevery country in the world now has a nationally organized influenzamonitoring system. For this scheme general practioners remove swabs fromthe nose and/or throat and send them to the respective nationalreference centre. The influenza A/B viruses are then usually detected byeluting the swabs and subsequently culturing the patient specimens onmammalian cells such as MDCK cells (Madine-Darby Canine Kidney cells).

The culture in these special laboratories can take up to 14 days and isthus not of immediate relevance for the diagnosis of the individualpatients. Rather the goal of the national reference centres is to typeand subtype the cultured viruses and to report the results to the WorldHealth Organisation (WHO). The job of the vaccine manufacturer is thento adapt next year's influenza vaccines to the latest circulating viralstrains on the basis of the annual WHO recommendation.

The reason for the high degree of genetic and hence immunologicalvariability especially of the influenza A viruses is due to the factthat a genetic shift (reassortment of the viral genes) can also occur inrare cases in addition to the usual genetic drift (point mutation). Thisis due to the fact that, in contrast to other viruses, the genome of theinfluenza viruses is segmented and that influenza A is a pathogen inhumans as well as in animals.

The immunodominant antigens present on the surface of the virus arehaemagglutin (H) and neuraminidase (N). At present 15 subtypes ofhaemagglutin (H1-H15) and 9 subtypes of neuraminidase (N1-N9) are knownfor influenza A.

If for example a host (e.g. a pig) is coinfected with an influenza virusof the A type which is pathogenic for man and with an influenza A birdvirus this can result in a reassortment of the viral genome to form anew influenza A virus subtype which then completely evades the humanimmune system when it is transferred back to humans. The most recentexample of this was the occurrence of the so-called avian virusinfluenza in May 1997 in Hong Kong (type A/H5N1/Hong Kong/156/97) inwhich 6 of the 18 affected patients died despite an early detection andintensive medical care.

However, in order to cause a larger epidemic or even a worldwidepandemic, a new influenza virus subtype must be transmitted directlyfrom human to human as was the case in 1918/1919 for the firstoccurrence of the subtype H1N1 (Spanish flu, worldwide ca. 50 milliondeaths), 1957 (H2N2, Asian flu, ca. 1 million victims) and 1968 (H3N2Hong Kong flu, ca. 1 million victims).

A new generation of influenza agents has recently become available, theso-called neuraminidase inhibitors which for the first time allow acausal treatment of influenza and are thus regarded among experts as atrue breakthrough in the treatment of influenza. Clinical studiescarried out for the registration of this new class of substance showedthat successful treatment primarily depends on an early start totreatment after the first clinical symptoms occur. Hence in view of thisnew therapeutic option, the necessity to begin the treatment at an earlystage and the relatively unspecific clinical symptoms, a rapidindividual diagnosis would be helpful as a basis for treatmentdecisions.

As a result several diagnostic manufacturers have recently developedrapid immunological influenza tests based on antigen detection and theserapid tests are nasal and/or throat swabs or liquid obtained by nasalirrigation as the sample material. Examples of such rapid influenzatests are the Influenza A/B Rapid Test from Roche Diagnostics GmbH,Quick Vue® from Quidel and AB FLU OLA® from Biostar.

A particular problem in collecting samples is to remove sample materialfrom the affected region of the nasal and pharyngeal cavity whichcontains sufficient amounts of influenza A and/or influenza B viruses(abbreviated to influenza A/B viruses in the following). Hence thequality of sampling has a direct influence on the positivity rate of therapid tests which usually have a clinical sensitivity of about 70% i.e.the immunological rapid test is also positive in 70% of all samples thatare assessed to be influenza positive by the reference method (cellculture). Hence the diagnostic manufacturers point out in the packageinserts for their influenza rapid tests that the specimens should onlybe collected by specially trained medical personnel in order to ensurethat the specimen material obtained does in fact contain adequateamounts of influenza A/B viruses.

The situation is further complicated by the fact that the regions in thepharyngeal cavity that have to be swabbed (posterior pharyngeal wall,pharynx, tonsils) can indeed differ depending on the viral infectionthat is present. This for example means that a swab has to be taken ofother regions of the throat in order to detect a streptococcal infectionthan for example to detect influenza A/B viruses.

Taking a nose/throat swab is unpleasant for patients and a particularproblem in the case of (small) children. On the other hand children arethe main carriers of virus influenza at least in the early phase of aninfluenza epidemic due to their social contacts (kindergarten, school)and the fact that their immune system is often not yet fully developed.

Since nose and throat swabs are not a homogeneous sample material, thepositivity rate of an influenza test is, in addition to the quality ofthe swab, also determined by the correct elution i.e. the transfer ofthe swab material to a liquid phase which then serves as the actualsample material in the subsequent test.

Especially the new therapeutic option (neuraminidase inhibitors) will infuture result in an increasing need for flu virus tests (influenza A/Bviruses) for general practitioners or the patients which is notcompatible with the previously used sample material (swab, nasalirrigation) that requires that specimens be taken by trained (medical)personnel.

The object of the present invention is to eliminate the disadvantages ofthe prior art. In particular the object of the invention is to provide amethod for detecting an influenza A and/or influenza B virus infectionwhich can be carried out by untrained personnel or ideally by thepatients themselves. Above all it is an object of the invention to finda sample material from which influenza A and/or influenza B viruses canbe reliably detected which is preferably homogeneous and can becollected by untrained personnel or ideally by the patient themselves ina simple and uncomplicated manner.

SUMMARY OF THE INVENTION

The invention concerns a method for the detection of an infection withthe influenza A and/or influenza B virus comprising the steps

i) obtaining a saliva sample from the individual to be examined

ii) preparing the saliva sample for the detection or a detectionreaction and

iii) detecting the influenza A and/or influenza B virus in the salivasample.

The invention additionally concerns a test kit for detecting aninfection with the influenza A and/or influenza B virus containing

i) a device for collecting a saliva sample and

ii) reagents and auxiliary agents for the detection of influenza Aand/or influenza B viruses.

Finally the invention concerns the use of saliva as a sample material todetect an infection with the influenza A and/or influenza B virus.

DETAILED DESCRIPTION

It was surprisingly found that it is possible to reliably detectinfluenza A/B viruses from saliva as a sample material using establisheddiagnostic methods.

In particular it is surprising that in a preferred embodiment of theinvention it is possible to detect influenza A/B viruses from salivawithout having to enrich the viruses in the saliva sample when taking asample, but rather spontaneously formed saliva is adequate as a samplematerial for detecting influenza A/B viruses. Spontaneously formedsaliva is understood in the present invention to mean that in order todetect influenza A/B viruses, the influenza A/B viruses are not enrichedin the sample when the saliva sample is collected. Only the saliva thatis present spontaneously in the mouth cavity is collected and forexample transferred to a saliva collecting vessel (e.g. by spittingetc.). It is also possible to collect the saliva sample in the mouthcavity with the aid of an absorbent material such as a cotton fleece(swab) or to use saliva collecting devices familiar to a person skilledin the art (such as the so-called SALIVETTES® from Sarstedt, Newton,N.C., or the ORASURE® specimen collection device from Epitope Inc.Beaverton, Oreg.) to collect the saliva sample.

Of course it is also possible according to the invention to use salivaas a sample material for the detection of influenza A/B viruses in whichan enrichment (concentration) of the viruses has occurred during thesampling or in subsequent processing steps.

In contrast to the previously known sample materials in influenzadiagnostics i.e. nose/throat swabs and liquid obtained by nasalirrigation, the saliva sample material according to the invention andthe simple manner of sample collection allows, a standardized samplecollection by untrained persons or by the patients themselves and thusincreases the diagnostic reliability of influenza A/B virus tests.Moreover saliva is a more homogeneous sample material than the otherpreviously used materials which also contributes to the diagnosticreliability.

In the method according to the invention a saliva sample is firstlycollected. This can for example be carried out as already describedabove by spitting into a vessel, absorbing a saliva sample by means ofan absorbent material such as a cotton swab or similar materials in theoral cavity or by using a conventional saliva collecting device.

The saliva sample is subsequently prepared for the detection reaction inaccordance with the detection method to be used (immunological detectionor detection by means of nucleic acids).

For the immunological detection the viral nucleoprotein of influenza A/Bviruses is for example released from the saliva sample by lysingreagents. Such reagents are known to a person skilled in the art and canfor example contain salts or detergents as the active components for thelysis. Lysing reagents for the detection of influenza viruses preferablycontain a detergent (for example TRITON®X100, TWEEN®20 orbeta-octylglycopyranoside have proven to be suitable), a mild reducingagent (for example N-acetyl L-cysteine or DTT=dithiothreitol),physiological saline (i.e. 0.9% by weight NaCl in 20-50 mM phosphatebuffer), a preservative (for example 0.09% by weight NaN₃) andoptionally a protein to reduce unspecific binding (for exampleBSA=bovine serum albumin or BPLA=bovine plasma albumin). In addition tothe viral nucleoprotein, the immunological test can for example alsodetect the viral matrix protein or the viral polymerases. It is alsopossible to detect the haemagglutinin or the neuraminidase of theinfluenza A/B viruses in which case no lysis is required since theseviral components are present on the viral surface.

If the detection is based on a nucleic acid, a viral nucleic acid (RNA)is for example isolated, purified and appropriately amplified in thepresence of the primers required for the detection, preferably by meansof the reverse transcriptase polymerase chain reaction (RT-PCR). Thesesteps are known to a person skilled in the art as are nucleic acid testswithout prior amplification.

The influenza A/B viruses are detected in the saliva sample by knownmethods.

In the case of an immunological detection of for example the viralnucleoprotein, a sandwich complex can be formed using appropriatelylabelled antibodies and detected. Competitive test formats are of coursealso possible. Although the detection is preferably by means of animmunochromatographic test strip that can be visually evaluated as arapid test, the detection can be carried out by means of allconventional immunological methods, for example ELISA, agglutinationtests, turbidimetric tests etc.

If the influenza A/B viruses are detected by means of nucleic acids suchas RNA, the product of the RT-PCR is preferably labelled by means of alabelled primer and the label on the primer (e.g. an enzyme orfluorescent label) is detected according to the type of label.

A test kit for carrying out the method according to the invention isalso a subject matter of the invention. The test kit contains as animportant component firstly a device for collecting a saliva sample suchas a vessel for spitting into, one or more absorbent cotton swabs or aconventional saliva collection device e.g. a SALIVETTE® device from theSarstedt, Inc, or an ORASURE® specimen collection device from theEpitope Inc. In addition the test kit contains all reagents andauxiliary reagents required to detect the influenza A/B viruses in thesaliva sample. In the case of an immunological detection, these are forexample a lysing buffer to release the viral nucleoproteins, optionallylabelled antibodies and optionally a reaction medium (test strips,microtitre plate, test tubes) for carrying out the immunologicaldetection. The corresponding reagents and auxiliary agents are known toa person skilled in the art in numerous embodiments. The same applies tothe nucleic acid test. In this case the kit for example contains therequired PCR reagents and reaction vessels.

The invention is further elucidated by the following example:

EXAMPLE 1. Detection of an Influenza Virus Infection from Saliva as theSample Material

In order to demonstrate the suitability of saliva as a sample materialfor detecting influenza viruses, two throat swabs and one saliva samplewere taken from 10 persons suspected to have an influenza infection and4 persons which were not suspected of having influenza. The samplematerial obtained from the throat swabs was collected for comparativepurposes and examined for an influenza A/B virus infection. The resultsof these investigations were compared with the results obtained usingsaliva as the sample material. Table 1 which is shown at the end of theexperimental part of the example shows an overview of the comparativeresults.

1.1 Sample Collection

1.1.1. Throat Swabs

The throat swabs were taken using sterile disposable cotton swabs fromthe Copan Italia S.p.a. (Brescia, Italy; order No. 167CS01) in themanner familiar to a person skilled in the art. The two throat swabstaken from each patient were obtained by swabbing the throat once with aswab having two cotton pads located directly adjacent to one another.This ensures that the two swabs are substantially comparable.

1.1.2. Saliva Sample

The saliva sample was collected by the patients themselves by collectingca. 0.5 ml spontaneously formed saliva in a small disposable plastictube with a screw cap (order No. 62.559.001 from the Sarstedt Inc.). Thesaliva collecting methods and devices known to a person skilled in theart (such as SALIVETTES® from the Sarstedt Inc. of the ORASURE® specimencollection device from Epitope Inc.) were intentionally not used inorder to demonstrate that it is possible to detect influenza viruseswithout prior concentration or saliva processing. Of course it isequally possible according to the invention to use such salivacollecting methods and devices.

1.2 Detection of Influenza A/B Viruses from Throat Swabs (Only forComparison)

1.2.1 Detection from Throat Swabs by Means of Cell Culture

The first throat swab in each case was immediately transferred aftercollection to a tube containing 1.5 ml influenza virus transport mediumfrom Virotest GmbH (Stuttgart, Germany, Cat. No. 0500300) and culturedon MDCK cells (Madine-Darby Canine Kidney cells). The samples werecultured according to the methods described in the literature(“Mikrobiologische Diagnostik”, Georg Thicme editor, Stuttgart, NewYork, 1992, publisher Friedrich Burkhard, page 371). The results areshown in table 1 in which “+” represents a positive result and “−”represents a negative result.

1.2.2 Detection from the Throat Swab by Means of an Immunological RapidTest

The second swab was in each case examined immediately after removing thesample from the throat of the patient by means of an immunologicalantigen rapid test for the presence of influenza viruses (Influenza A/BRapid Test from Roche Diagnostics GmbH, Mannheim, Germany, cat. No. 2158 663). The rapid test used in this case corresponds essentially tothe immunological rapid test described as example 2 in EP-A 0 926 498.Reference is herewith expressly made to this document.

In addition to the visual evaluation of the test strips, the intensityof the detection line after completion of the chromatography wasquantitatively measured by means of a remission photometric instrument(ring illumination using 24 green LEDs at a wavelength of 555 nm and CCDcamera with a lens). The intensity of the detection line signal wasexamined as a percentage of the remission (% rem; relative to a “white”area of the test strip which was assigned a remission of 100%):remission values above 98.5% are detected as a negative signal by theuser; remission values between 96% and 98.5% are detected as a weaklypositive signal; remission values of less than 96% are detected as anunequivocally positive signal. The results are shown in table 1 in which“+” denotes a positive signal, “(+)” denotes a weakly positive signaland “−” denotes a negative signal.

1.3 Detection of Influenza A/B Viruses from Saliva

1.3.1. Detection of Viral Nucleic Acid in the Saliva by Means of RT-PCR

The saliva samples were examined by means of RT-PCR (reversedtranscriptase polymerase chain reaction) for the presence of viralinfluenza nucleic acid (in this case ribonucleic acid, RNA). Theanalytical process comprises the three conventional partial steps innucleic acid diagnostics of sample preparation (in order to separateinhibitors), amplification and detection of the nucleic acid (cf.1.3.1.a to 1.3.1.c in the following). The procedure was as follows:

1.3.1.a Sample Preparation

The commercially available High Pure Viral RNA Kit from Roche MolecularBio-chemicals (Mannheim, Germany; order No. 1 858 882) was used toisolate the viral influenza RNA. The procedure was according to thestandard protocol described in the product description by firstlybinding 200 μl saliva per reaction vessel (filter tube) to the glassfleece of the filter tube in the presence of binding buffer and, afterremoving inhibitors that may be present by two wash steps, eluting theviral nucleic acid in 200 μl elution buffer. In this process care wastaken that the sample volume before (saliva) and after (eluate) thesample preparation was identical so that no concentration of theinfluenza viruses had taken place. Of course, it is also possibleaccording to the invention to carry out the elution step with a smalleramount of elution buffer than the quantity of saliva and thus toconcentrate or enrich the influenza viruses in the eluate.

1.3.1.b RT-PCR

If not listed otherwise all reagents were obtained from Roche MolecularBiochemicals (see above).

The reaction volume per PCR reaction vessel was 50 μl. This contained 10μl sample volume (eluate from the sample preparation) and 10 μl bicinebuffer (5×RT-PCR buffer). The other components of the master mix werepresent in the following final concentration: 2.5 mmol/l manganeseacetate, 0.2 mmol/l of each of the following dATP (2′-deoxyadenosine-5′triphosphate), dCTP (2′deoxycytidine-5′ triphosphate), dGTP(2′-deoxyguanosine-5′ triphosphate) and dUTP (2′-deoxyuridine-5tri-phosphate) and 0.05 mmol/l dTTP (2′-deoxythymidine-5′ triphosphate);0.01 U/μl UNG (uracil DNA glycosylase); 0.2 U/μl Tth polymerase fromThermus thermophilus HB8; 0.8 U/μl RNase inhibitor and 1.0 μmol/l offorward and reverse primer.

The sequence of the primers was obtained from the literature (James C.Donofrio et al., Detection of Influenza A and B in RespiratorySecretions with the PCR, 1992, PCR Methods and Applications 1, page263-268). The primers are type-specific for influenza A and have ahighly conserved gene segment (position 101 to 312) of the matrix geneof 212 base pairs in length. The reverse primer was labelled at the 5′end with biotin for subsequent detection of the amplification product inthe microtitre plate. Type-specific primers for influenza B are alsoknown in the literature (James C. Donofrio et al., Detection ofInfluenza A and B in Respiratory Secretions with the PCR, 1992, PCRMethods and Applications 1, page 263-268).

The master mix was amplified in a Perkin Elmer 9600 thermocycler usingthe following temperature profile: 20 minutes room temperature afteraddition of UNG+45 minutes 60° C.+2 minutes 94° C.+10 cycles (30 seconds94° C.+60 seconds 50° C.+90 seconds 68° C.)+35 cycles (30 seconds 94°C.+60 seconds 60° C.+90 seconds 68° C.)+7 minutes 68° C.

1.3.1c Hybridization and Detection

The nucleic acid amplified by the RT-PCR was detected using the PCRELISA (DIG detection) from Roche Molecular Biochemicals cat. No. 1 636111. With the exception of the pipetting volumes, all steps were carriedout according to the package insert.

10 μl of the amplification product was mixed with 20 μl denaturatingsolution in a 1.5 ml reaction vessel and incubated for 10 minutes atroom temperature. Afterwards 250 μl of a digoxigenin-labelledhybridization probe was added. The concentration of the hybridizationprobe was 70 ng/ml.

The sequence of the hybridization probe was obtained from the literature(James C. Donofrio et al., Detection of Influenza A and B in RespiratorySecretions with the PCR, 1992, PCR Methods and Applications 1, page263-268). The probe is directed against position 177-205 of the matrixgene (segment 7) of influenza A. Corresponding probes for influenza Bare also known from the literature (James C. Donofrio et al., Detectionof Influenza A and B in Respiratory Secretions with the PCR, 1992, PCRMethods and Applications 1, page 263-268).

200 μl of the reaction mixture was transferred to a well of astreptavidin-coated microtitre plate and incubated on a shaker for 1hour at 37° C. After binding the hybridization product to thestreptavidin of the microtitre plate wall, the contents of the wellswere aspirated and washed three times with 300 μl wash solution eachtime. Afterwards 200 μl anti-digoxigenin-peroxidase substrate(anti-DIG-POD conjugate) was added to the well and the solution wasincubated for 30 minutes at 37° C. on a shaker. After binding the PODconjugate the contents of the wells were aspirated, washed 3 times with300 μl wash solution and subsequently 200 μl2,2′-azino-di-[3-ethylbenzthiazoline sulfonate (6)] diammonium saltsubstrate (ABTS substrate) was added. After a final incubation for 30minutes at 37° C., the colour development at 405 nm was measured on amicrotitre plate reader.

For each PCR determination of saliva samples, several negative controls(DEMC water) (article No. 16-001Y, ACCUGENE® Water, molecular biologygrade, autoclaved, manufactured by Cambrex Bio Science VerviersS.P.R.L., Verviers, Belgium) were carried out in parallel to excludefalse-positive values by contamination and several positive controls(dilution series of MDCK culture supernatant containing influenzaviruses) were determined in parallel by sample preparation and RT-PCR.In addition an internal kit positive control was also carried out inorder to check the detection reagents on the microtitre plate.

The absorbance values of the parallel negative controls in themicrotitre plate were usually 100-150 mA. The values of the internalpositive control of the kit that was carried out in parallel wereusually 1000-1500 mA.

Absorbance values of =300 mA were defined as a positive signal forpatient samples which corresponds to a signal that was more than twicethe blank or zero value. The results are shown in table 1 in which “+”denotes a positive result and “−” denotes a negative result.

1.3.2. Detection of a Viral Antigen in Saliva by Means of anImmunological Rapid Test

The Influenza A/B Rapid Test from Roche Diagnostics GmbH (cat. No. 2 158663) was used to detect influenza A/B viruses in saliva. The rapid testthat was used corresponds essentially to the immunological rapid testdescribed as example 2 in EP-A 0 926 498. Reference is expressly madeherewith to this document.

The test is usually used to type-specifically detect viral nucleoproteinin throat swabs by means of an immunological chromatographic test strip.The test does not differentiate between influenza A and influenza Bviruses.

In order to ensure a good chromatography of the test strip using salivaas the sample material, a special lysing/elution buffer was used whichis not a component of the test kit. The special lysing/elution bufferhas the following composition:

0.9% by weight NaCl, 2 mM KH₂PO₄, 10 mM Na₂HPO₄, 0.095% by weight NaN₃,10 mM EDTA, 1.5% by weight bovine serum albumin (BSA), 1.5% by weightTriton®X-100.

The test was carried out as follows:

Instead of firstly eluting a throat swab as the patient sample with 3portions (=800 μl) of the lysing/elution buffer contained in the testkit as described in the package insert, 300 μl saliva and 500 μl of thespecial lysing/elution buffer were added to a reaction vessel and mixed.

The remaining test procedure was carried out according to the stepsdescribed in the package insert. This comprises firstly adding two dropsof antibody solution 1 (contains biotinylated monoclonal antibodiesagainst nucleoprotein A and nucleoprotein B), adding two drops ofantibody solution 2 (contains digoxigenylated monoclonal antibodiesagainst nucleoprotein A and nucleoprotein B) and subsequentlychromatographing this reaction mixture on a test strip.

As known from EP-A 0 926 498 the test strip contains a conjugate fleecewhich is reversibly impregnated with a gold conjugate that can bedetached in the sample liquid. The gold particles are adsorptivelycoated with a monoclonal antibody against digoxigenin.

A nitrocellulose membrane on which polystreptavidin as a detection lineand a polyclonal antibody PAB<mouse Fcγ>S-IgG as a control line areirreversibly impregnated is located in another zone of the test stripdownstream in the direction of chromatography.

The sandwich complex formed in the reaction vessel in the presence ofthe analyte comprising biotinylatedantibody/nuleoprotein/digoxigenylated antibody chromatographs across thetest strip, binds the gold conjugate after it has been solubilized viathe anti-digoxigenin antibody to the digoxigenin-labelled anti-influenzaantibody of the sandwich complex and is subsequently captured on thepolystreptavidin line on the nitrocellulose membrane by means of thebiotin-labelled anti-influenza antibody of the sandwich complex. As aresult a red line becomes visible on the nitrocellulose membrane whichrepresents a positive test signal.

Excess gold conjugate chromatrographs downstream and is captured on thecontrol line of the nitrocellulose membrane as another visible red lineby means of PAB<mouse Fcγ>S-IgG.

In addition to the visual evaluation of the test strips, the intensityof the detection line after completion of the chromatography wasquantitatively measured by means of a remission photometric instrument(ring illumination using 24 green LEDs at a wavelength of 555 nm and CCDcamera with a lens). The intensity of the detection line signal wasexamined as a percentage of the remission (% rem; relative to a “white”area of the test strip which was assigned a remission of 100%);remission values above 98.5% are detected as a negative signal by theuser; remission values between 96% and 98.5% are detected as a weaklypositive signal; remission values of less than 96% are detected as anunequivocally positive signal. The results are shown in table 1 in which“+” denotes a positive signal, “(+)” denotes a weakly positive signaland “−” denotes a negative signal.

1.4. Results

Some results obtained with patient samples are shown in the following asan example (Table 1).

These results relate exclusively to the detection of influenza A virusessince only influenza A was predominant at the time of sample collectionand no patient samples with influenza B were found. Experts know thatinfluenza B, in contrast to influenza A, does not circulate in everywinter season and even if both influenza A/B viruses occur in a winterseason, influenza B is always much less prevalent.

It should also be mentioned that artificially prepared influenzaB-positive saliva samples (pooled saliva samples which have been spikedwith a culture supernatant of influenza B viruses) were also suitablefor detecting influenza viruses in saliva in the nucleic acid test aswell as in the immunological test. The results shown do not thereforerepresent a limitation in the sense of the invention with regard toseparate or joint detection of influenza B viruses in addition toinfluenza A viruses from saliva. Rather saliva is suitable as a samplematerial for the detection of influenza A/B viruses.

TABLE 1 Throat swab samples Saliva samples (only for comparison)immunological immunological rapid test Person rapid test PCR photo-No.^(A) culture^(B) visual^(C) photometric^(D) result^(E) visual^(C)metric^(D) 1 + + + + + + 2 + (+) (+) + + + 3 + (+) (+) + + +4 + + + + + + 5 + + + + + + 6 + (+) (+) + + + 7 + − − + (+) (+) 8 + −− + (+) (+) 9 − − − − − − 10 − − − − − − 11 − − − − − − 12 − − − − − −13 − − − − − − 14 − − − − − − ^(A)Persons No. 1-10 were persons withinfluenza-like symptoms. Persons No. 11-14 were persons without acuterespiratory symptoms. ^(B)Culture on MDCK cells (cf. 1.2.1 above); +positive; − negative ^(C)Rapid test procedure as described under 1.2.2.and 1.3.2. (see above); + positive; (+) weakly positive; − negative^(D)Quantitative determination of the intensity of the detection line bymeans of a remission-photometric measuring instrument (cf. under 1.2.2.and 1.3.2. above); only the relative results are shown (+ positive; (+)weakly positive; − negative) which are according to the scale given inthe text. ^(E)The absorbance values of the microtitre plate weremeasured at 405 nm (cf. under 1.3.1. above); only the relative resultsare shown (+ positive; − negative) which are according to the scalegiven in the text.

The following is apparent from the results shown in Table 1:

Not all persons (No. 1-10) classified as influenza patients according tothe clinical symptoms were in fact influenza positive. Persons No. 9 and10 were negative in the culture of the throat swab as well as in the PCRof the saliva as well as in the rapid test of the saliva and throatswab. This emphasizes the statement made in the introduction and knownamong experts that it is not possible to make a definitive diagnosis forinfluenza solely on the basis of clinical symptoms.

The throat swabs and saliva samples of asymptomatic persons (No. 11-14)were negative with all listed test methods which demonstrates theclinical specificity of these methods.

Of the eight persons found to be influenza A positive by means of cellculture by throat swabs (No. 1-8; so-called culture-positive persons),six persons were also diagnosed as positive with the immunological rapidtest using throat swabs. This illustrates that the clinical sensitivityof these previously available rapid tests using swabs as a samplematerial is less than that of the cell culture which is recognized atpresent as the gold standard (clinical sensitivities of variousimmunological rapid tests according to the package inserts of therespective manufacturers: Quidel 73% for nose swabs, Roche 70% forthroat swabs, Biostar 60% for throat swabs and 83% for nasopharyngealswabs).

In contrast the corresponding saliva samples from all eightculture-positive patients were found to be positive with theimmunological rapid test.

Furthermore the comparison of the remission values (not shown in thetable) shows that the intensities of the detection line on theimmunological rapid test strips using saliva samples were somewhat moreintensive than the corresponding intensities of the detection line whenusing throat swabs.

The results of the rapid tests of saliva are thus in agreement with thecorresponding PCR results from saliva and demonstrate that influenza A/Bviruses can be reliably detected in saliva as a sample material evenwithout prior enrichment during the sample collection.

What is claimed is:
 1. A method for determining whether a subject isinfected with an influenza A virus and/or an influenza B viruscomprising detecting at least one of influenza A virus or influenza Bvirus in a saliva sample from the subject.
 2. The method of claim 1,wherein the saliva sample is obtained from spontaneously formed saliva.3. The method of claim 1, wherein the saliva sample is obtained with asaliva collection device without virus-specific enrichment.
 4. Themethod of claim 1 further comprising isolating at least one of viralinfluenza A RNA and viral influenza B RNA from the sample.
 5. The methodof claim 4 wherein the detecting comprises amplifying the RNA by RT-PCRand detecting the amplified RNA.
 6. The method of claim 1 wherein thedetecting comprises examining the saliva sample immunologically for thepresence of at least one of the influenza A virus and the influenza Bvirus.
 7. The method of claim 6 further comprising treating the samplewith a lysing buffer to release a viral nucleoprotein of at least one ofthe influenza A virus and influenza B virus.
 8. The method of claim 7wherein the nucleoprotein is detected immunologically.
 9. A method fordetecting in a subject an infection with influenza A virus and/orinfluenza B virus comprising: (a) obtaining a saliva sample from thesubject; (b) treating the sample with a lysing agent to release viralnucleoprotein of at least one of influenza A virus and influenza Bvirus; (c) contacting the treated sample with at least one antibody tothe at least one viral nucleoprotein; and (d) detecting whether the atleast one antibody binds to the at least one viral nucleoprotein,thereby detecting an infection with at least one of influenza A virusand influenza B virus.
 10. The method of claim 9, wherein the salivasample is obtained from spontaneously formed saliva.
 11. The method ofclaim 9, wherein the saliva sample is obtained with a saliva collectiondevice without virus-specific enrichment.
 12. A method for detecting aninfection with influenza A virus and/or influenza B virus comprising:(a) obtaining a saliva sample; (b) amplifying viral RNA in the sample byRT-PCR; (c) contacting the amplified RNA with at least one hybridizationprobe for the viral RNA of at least one of influenza A virus andinfluenza B virus; and (d) detecting whether the at least one probehybridizes to the amplified RNA, thereby detecting an infection with atleast one of influenza A virus and influenza B virus.
 13. The method ofclaim 12, wherein the saliva sample is obtained from spontaneouslyformed saliva.
 14. The method of claim 12, wherein the saliva sample isobtained with a saliva collection device without virus-specificenrichment.
 15. A test kit for the detection of an infection withinfluenza A virus and/or influenza B virus comprising: (a) a device forcollecting a saliva sample, and (b) reagents for detecting at least oneof influenza A virus and influenza B virus in the saliva sample.
 16. Thekit of claim 15 wherein the reagents are suitable for detecting thenucleic acid of at least one of influenza A virus and influenza B virus.17. The kit of claim 15 wherein the reagents are suitable forimmunologically detecting the nucleoprotein of at least one of influenzaA virus and influenza B virus.
 18. The kit of claim 15 wherein thedevice for collecting a saliva sample is suitable for collectingspontaneously formed saliva.